BIO230 Lecture 4 Flashcards
Different RNA transcribed by different _________ in eukaryotes
RNA Pol
List some different types of RNA that cells produce.
mRNA
rRNA
tRNA
snRNA (small nuclear - nuclear processes, splicing of pre-mRNA)
snoRNA(small nucleolar - modify rRNA)
scaRNA (small cajal - modify snoRNA & snRNA)
miRNA (micro - regulate gene expression, block transcription of certain mRNA)
siRNA (small interfering - turn off gene expression)
non-coding RNA
Different types of RNA Pol only available in prokaryotes or eukaryotes?
Eukaryotes; prokaryotes have a single type of RNA Pol
What are the different different RNA Pol in eukaryotes?
RNA Pol I = rRNA genes
RNA Pol II = protein coding genes, snoRNA, miRNA
RNA Pol III = tRNA, rRNA, snRNA, other small RNA
What are general transcription factors and where are they found?
Found in eukaryotes
Help position RNA Pol at eukaryote promotors
Transcription initiation in eukaryotes requires many…
general transcription factors
What are the transcription factors needed by RNA Pol II to initiate transcription?
TFIID (TBP, TAF) TFIIB TFIIF TFIIE TFIIH
(_B_DEF_H)
What does the subunits of the TFIID do?
TBP - recognizes TATA box
TAF - recognizes other DNA sequences near transcription start site
What does TFIIB do?
Recognizes BRE element in promoter; positions RNA Pol at start site
What does TFIIF do?
Stabilizes RNA Pol interaction with TBP & TFIIB
Attract TFIIE and TFIIH
What does TFIIE do?
attracts & regulates TFIIH
What does TFIIH do?
Unwind DNA at transcription start point
Phosphorylates Ser5 of RNA Pol CTD
Release RNA Pol from promotor
Describe eukaryote gene regulation in detail.
- RNA Pol II transcribes protein coding genes
2. Need transcription factors TFIIB, TFIID, TFIIE, TFIIF, TFIIH
That “transcription factor” does prokaryotes need?
stigma factor
Where is the TATA box?
downstream of the genetic material (close though); small green box on diagram
What is the mediator?
An intermediate between regulatory proteins and RNA Pol
Holds all the transcription factors together (so many!) and allows activator to activate everything at once.
What are two additional factors that bind to RNA Pol besides transcription factors?
- Chromatin remodelling complex
2. Histone-modifying enzyme
How is eukaryotic gene expression controlled?
many regulatory proteins - activators and repressors
About how many eukaryotic proteins are encoded by the human genome?
~2000
How far can gene regulatory proteins act?
short, or VERY large distances (>10000 base pairs away)
What is the traditional definition of a gene?
coded “gene” region + promotor + regulatory proteins
What is the modern definition of a gene?
only the part that gets transcribed; does not include regulators
Where do gene regulatory proteins go to affect transcription?
- Mediator
- TF and RNA Pol
- chromatin structure of DNA control region
What is the same for all RNA Pol II? What is different for every gene?
Same: mediator, general transcriptional factors
Different for every gene: regulatory proteins + their binding site locations
What to gene regulatory proteins affect?
rate of transcription initiation
What does the regulatory sequence serve as?
binding sites for regulatory proteins.
What does the gene control region for gene X in eukaryotes consist of?
- Regulatory sequence
- gene regulatory proteins
- spacer DNA
- promotor: general transcription factors, TAT box, RNA Pol II
- gene X
Eukaryotic gene regulatory proteins often function as _____ on DNA
Protein complexes
In solution, gene regulatory proteins are…
separate, not attached and free floating
On DNA, gene regulatory proteins…
Bind to appropriate spots (gene regulatory sequences) and come together as a protein complex
What are coactivators and corepressors and what do they do?
Coactivators activate transcription when bound.
Co repressors repress transcription when bound.
They bind to DNA-bound regulatory proteins; DO NOT BIND DIRECTLY TO DNA!
What does DB and AD stand for?
DNA binding domains
Activation domain
What does the DNA binding domain do?
Recognizes specific DNA sequences
What does the activation domain do?
Accelerates rate of transcription.
On a protein, where is the DB and the AD relative to each other?
AD is on the top, BD is on the bottom (to bind to the DNA)
Eukaryotic activator proteins have an _____ design that allows scientists to mix and match ____________.
Modular
BD and AD
An example of a modular design eukaryotic activator protein is…
Gal4 gene used to activates transcription of yeast genes that code for enzymes to convert galactose to glucose
How does the Gal4 gene work when mixed and matched?
Normally, the Gal4 BD recognizes the recognition sequence for Gal4 and this will allow the activator protein to bind to the TATA box and turn LacZ gene transcription on.
Chimeric Gal4-LexA activator protein will not recognize the recognition sequence for Gal4. Nothing will be activated and LacZ gene is off.
Chimeric Gal4-LexA will recognize recognitions sequence for LexA, then turn LacZ gene on.
LacZ is a reporter gene in this instance.
What is a reporter gene?
Reports activity of gene control regulation.
How do activator proteins activate transcription?
- Attract
- Position
- Modify
General transcription factors
Mediator
RNA Pol II
Can be done…
Directly by acting on above components
Indirectly by modifying chromatin structure
How does direct eukaryotic gene regulation work?
Activator proteins bind directly to transcriptional regulators or mediator
Attract transcriptional regulators / mediator to promotors
What is direct eukaryotic gene regulation similar to?
Prokaryotic activators
How does indirect eukaryotic gene regulation work?
Activator proteins alter chromatin structure
What is a nucleosome?
- Basic structure of eukaryotic chromatin.
- DNA around a histone octamer
- ~200 nucleotide pairs of DNA
- core histone and the string on its left
What is a histone octamer composed of?
H2A, H2B, H3, H4 <- 2 of each makes 8 :)
What is linker DNA?
The string of DNA nucleotides between two histone cores
How many nucleotide pairs are in one nucleosome?
~200
What do nucleosomes pack as?
Chromatin fibers
What are the two models of nucleosome packing?
Zigzag model and solenoid model
What is the correct model for nucleosome packing?
Either the zigzag model or the solenoid model…we aren’t sure.
What is one problem that transcriptional regulators face when confronted with tightly packed DNA?
Cannot assemble on promotors because it is so tightly packaged in packed nucleosomes
How do activator proteins alter chromosome structure and increase promoter accessibility?
- Chromatin remodelling complex
- Chromatin remodelling complex & histone chaperones -> histone removal
- Chromatin remodelling complex & histone chaperones -> histone replacement
- Histone modifying enzyme
Can be 1 or a combination of the 4.
Do the 4 methods that an activator protein use to increase accessibility of the promoter area work independently or together?
Can work independently, but often works together
What makes it easier for histone chaperones to remove histones from the nucleosome?
Prior acetylation of the histone
Nucleosomes structure can be altered by ___________ in an __________ manner to increase promotor accessibility .
Chromatin remodelling complexes
ATP-dependent
What is nucleosome sliding and how does it work?
A way for nucleosomes to be altered to make promoters more accessible.
Catalyzed by ATP dependent chromatin remodelling complexes.
Chromatin remodelling complex holds onto nucleosome and pushes DNA to loosen tents attachment to nucleosome core
ATP attachment, ATP hydrolysis, release of ADP and P = move DNA along to the right slightly
Many cycles needed to complete DNA sliding
What does nucleosome removal and histone exchange require cooperation with?
Histone chaperones
How does nucleosome removal work and why is it needed?
To make promoter region more accessible for transcription machinery.
Catalyze by ATP dependent chromatin remodelling complex.
ATP hydrolysis will completely remove the histone core.
Histone chaperone will bind to histone core and remove it.
End with DNA lacking nucleosome.
How does histone exchange work and why is it needed?
To make promoter region more accessible for transcription machinery.
Requires ATP dependent chromatin remodelling complex.
- Swap out half the nucleosome (H2A-H2B diamer) with a variant (H2AZ-H2B)
- After nucleosome removed, a new nucleosome core can be inserted
Both will bind to slightly different regions of DNA than the original.
What two nucleosome subunits are found as a diamer?
H2A-H2B
What does the histone-modifying enzyme do once it binds to the nucleosomes?
signal for chromatin remodeling
What do histone modifying enzymes produce?
specific patterns of histone modifications / code
what is another word for histone modification?
Histone code
What are 3 ways this that histone modifying enzymes modify histones?
- phosphorylation
- acetylation
- methylation
define: phosphorylation
addition of a phosphate group
define: acetylation
addition of a acetyl group
define: methylation
addition of a methyl group
Which enzyme is in charge of phosphorylation?
Kinase
Which enzyme is in charge of acetylation?
Acetyltransferase
Which enzyme is in charge of methylation?
Methyltransferase
Histone modifications occur on ________ of _______
Amino acids
Histones tails
What particular histone modifying complex is in charge of doing specific modifications to histone tails?
“Writers”
Write on histone tails; modify or add groups
How many histone tails are on a histone?
8; one for each subunit
Ex. 2 H2A tails, 2 H2B tails…
Are the histone tails the N terminal or the C terminal of the protein?
N terminal
Are all histone tails modified differently depending on what amino acid it it holds information for?
Yes
During modification of the histone tails, what factors affect the outcome of the modifications?
- where on the tail is being modified
- what does the modifying (phosphorylation, acetylation, methylation)
Why is the histone “code” a code?
Because the code is read by “readers”
What are reader proteins?
Proteins that can recognize specific modifications and provide meaning to the histone code
What are protein writers?
Modify the histone tails by adding or changing groups
What is one example of transcriptional regulation using the histone code?
human interferon gene promotor
How does the human interferon gene promotor work, and what is it an example of?
Example of transcription regulation using the histone code.
- activator protein bind to chromatin; attracts a histone acetyltransferase (HAT)
- HAT acetylates lysine 8 of H4 (H4K8), lysine 9 of H3 (H3K9) (4 things being acetylated)
- Activator attracts histone kinase (HK)
- HK phosphorylates serine 10 of H3 (only occur after acetylation of H3K9)
- Serine causes acetylation of H3K14 -> histone code for transcription initiation written
- TFIID & chromatin remodelling complex & remaining transcription machinery bind to acetylated histone tails; initiate transcription
How do we abbreviate lysine 7 of histone H2A?
H2AK7
What is the activator off the human inferring end promoter?
Gene activator protein
Where does the gene activator protein bind to?
DNA binding domain
What does HAT stand for?
Histone acetyltransferase
What would you see at the interferon gene promotor and accompanying histone if histone kinase was inhibited?
Acetylated H3K9 and H4K8
Nothing else because steps of writing the histone code is sequential; everything before HK is done, nothing after.
What is the differences between prokaryotes and eukaryotes in term of transcriptional repression?
Eukaryotic repressor proteins rarely compete with RNA pol for access to DNA
What are the ways that eukaryotic repressor proteins inhibit DNA transcription?
- Prevent activators from binding (binding site for repressor and activator overlap)
- Bind to activator surface to stop the activator from attracting RNA pol
- Bind to TFIID to prevent activator surface from reaching TFIID -> other TF cannot assemble
- Recruits chromatin remodelling complexes so it can’t bind to histones (cannot expose promoter region)
- Recruits histone deacetylase (works opposite of HAT, which promotes transcription initiation); remove acetyl groups, more compact histone structure prevents transcription
- Recruits histone methyl transferase: methylate histone tails -> attract proteins that maintain compact chromatin structure
What are histone reader and writer proteins guided by to establish a repressive form of chromatin?
gene regulatory proteins
How do gene regulatory proteins guide histone reader and writer proteins to establish a repressive form of chromatin? What does this prove?
- Gene regulatory protein that represses gene expression binds to chromatin
- Recruits histone modifying enzyme (writer) which modify the histone
- Reader protein comes and reads what is modified while writer modifies the next histone on the chain
- Another reader protein reads the next modified histone while writer moves one step forward again
- Process is repeated
- In the end, there is one reader on each histone, but only one writer protein
- Chromatin stabilized; no gene expression
Proves: histone code can spread!
Spreading of the histone code along chromatin is carried out by…
reader-writer complexes
after a histone is modified by a writer, what is left behind?
histone modification mark (small dot)
what is another word for histone modifying enzyme?
“writer”
what is another word for code-reader protein?
“reader”
How does the recruitment of code-reader-writer complexes spread histone code along chromatin?
- reader-writer complexes recruited by gene regulatory proteins to specific site on chromatin
- gene regulatory protein
- recruit writer
- writer recruit reader
- recruit writer on next histone
- every writer leaves a histone modification mark
- reader must recognize same mark that writer produces
What is the purpose of reader-writer complexes?
create specific modifications on nucleosomal histones
What do readers on chromatin attract?
DNA methylase
What does DNA methylase do on chromatin that is filled with reader-writer complexes?
methylates cytosine in DNA
What is the purpose of methylating cytosines on DNA?
to attract DNA methyl-binding proteins, which bind methyl groups and stabilize the whole chromatin structure (top is covered by reader-writer complexes, bottom by DNA methyl-binding proteins)
After DNA methylase methylates the cytosines on DNA, what happens?
DNA methyl binding proteins bind methyl groups to the methylated cytosines and stabilize the structure
after methyl groups are bound, what is the chromatin like?
very insulated; RNA Pol cannot get in
Gene expression patterns can be inherited. What is this called?
epigenetic inheritance
______ and therefore ______ can be inherited.
methylation
genetic expression patterns
With what we know from this lecture, what are the base pairs of DNA?
Adenine Tyrosine Guanine Uracil Cytosine Methylated cytosine
What is epigenetic inheritance?
ability of a daughter cell to retain memory from the parental gene expression pattern without any DNA sequence modification. (cell memory)
Vorinostat and Romidepsin are currently being used to treat curtaneous T cell lymphoma. These drugs inhibit enzymes that de-acetylate histones. Based on this knowledge, which statement regarding the behaviour of T cell lymphomas is most likely true?
a) Interferon is expressed at a high level in T cell lymphomas
b) T cell lymphomas have a more transcriptionally repressed transcriptome than regular cells.
c) the T cell lymphomas proteome should contain more proteins than normal
d) T cell lymphomas are more transcriptionally active than regular cells.
B
Normally, there are a lot of de-acetylate histones in T cell lymphomas (because drugs prevent de-acetylate; work in opposite way). De-acetylation means less transcription.
